Achieving the full potential of intraarterial, intravenous, or pulmonary gene therapy requires quantitative bioengineering analysis of extracellular and intracellular barriers. The central focus of this proposal is to achieve high expression of transgenes delivered to cardiovascular and cardiopulmonary cells such as arterial endothelium and airway epithelium. We will bioengineer and investigate sterol, peptide, and polymer- based transfection vehicles for their ability to enhance gene transfer. The following specific aims are proposed: Aim 1 Inflammatory Barriers: We will test a novel class of cationic glucocorticoids for enhanced plasmid and viral transfer and reduction of local inflammation in a mouse lung model. Preliminary data demonstrated a striking reduction of cellular infiltrate after adenovirus instillation. We will also explore the mechanism of anti-inflammatory action of cationic glucocorticoids on endothelial and white blood cell types challenged with various vectors used for gene therapy. Aim 2 Endosomal Barriers: Endosomal barriers include poor endosomal escape, endosomal degradation of plasmid, and Toll-like receptor 9 (TLR9) activation. We will test the effect of DNase II knockdown on subsequent lipofection and TLR9 activation in endothelium and pulmonary epithelium. Aim 3 Nuclear Barriers: Nuclear targeting in nondividing cells is often rate limiting. We have exploited the nonclassical nuclear localization sequence M9 derived from heteronuclear ribonuclear protein (hnRNP) A1 for transfection of endothelium, neurons, and mouse embryonic stem cells. Studies are proposed to explore nuclear targeting of oligonucleotides for repair of genomic DNA at specific sites. Aim 4 Unpackaging Barriers: Plasmid unpackaging after endosome escape is a difficult virus-like property to engineer. We present preliminary data for a cationic, crosslinkable polymer that protects DNA from nucleases and allows for light-triggered DNA release. Overall, these bioengineering studies will focus on barriers/rate limits that prevent high efficiency gene transfer.